Published Ahead of Print 3 October 2011.
10.1128/AAC.00521-11.
2011, 55(12):5936. DOI:
Antimicrob. Agents Chemother.
Narcisa Mandras, Janira Roana and Anna Maria Cuffini
Merlino, Daniela Scalas, Vivian Tullio, Giuseppe Garneri,
Valeria Allizond, Giuliana Banche, Franca Giacchino, Chiara
Caspofungin on Polymorphonuclear Cells
Transplant Recipients: Effect of
Candida albicans Infections in Renal
http://aac.asm.org/content/55/12/5936
Updated information and services can be found at:
These include:
REFERENCES
http://aac.asm.org/content/55/12/5936#ref-list-1
This article cites 23 articles, 7 of which can be accessed free at:
CONTENT ALERTS
more»
articles cite this article),
Receive: RSS Feeds, eTOCs, free email alerts (when new
http://aac.asm.org/site/misc/reprints.xhtml Information about commercial reprint orders:
http://journals.asm.org/site/subscriptions/ To subscribe to to another ASM Journal go to:
on November 18, 2011 by DIP DI SANITA PUBBLICA E MICROBIOLOGIA
http://aac.asm.org/
ANTIMICROBIALAGENTS ANDCHEMOTHERAPY, Dec. 2011, p. 5936–5938 Vol. 55, No. 12 0066-4804/11/$12.00 doi:10.1128/AAC.00521-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Candida albicans Infections in Renal Transplant Recipients: Effect of
Caspofungin on Polymorphonuclear Cells
䌤
Valeria Allizond,
1Giuliana Banche,
1Franca Giacchino,
2Chiara Merlino,
1Daniela Scalas,
1Vivian Tullio,
1Giuseppe Garneri,
2Narcisa Mandras,
1Janira Roana,
1and Anna Maria Cuffini
1*
Department of Public Health and Microbiology, University of Turin, Via Santena 9, 10126, Turin, Italy,1and Nephrology and
Dialysis Unit, Civil Hospital, Piazza della Credenza 2, 10015, Ivrea, Turin, Italy2
Received 18 April 2011/Returned for modification 12 July 2011/Accepted 23 September 2011
This study aimed to compare the caspofungin immunomodulating activities against Candida albicans on polymorphonuclear cells (PMNs) from renal transplant recipients (RTRs) and healthy subjects (HSs). RTR PMNs showed a significantly reduced fungicidal activity compared with that of HS PMNs. Addition of caspofungin to RTR PMNs significantly potentiated the yeast intracellular killing rate, achieving values similar to those observed for HS PMNs. These data show that caspofungin is suitable for invasive candidiasis treatment in patients with immune system-impaired components.
Renal transplant recipients (RTRs) are highly susceptible to invasive fungal infections (IFIs) mainly due to Candida spp., which continue to be the most frequent cause of death (60%) in the posttransplant period (1, 2, 13–15, 21). In fact, Candida spp. may easily find ideal conditions for growing and invading tissues in relation with both long-term immunosuppressive treatment and decreased defense mechanisms (1, 7).
Among newer antifungals, echinocandins such as caspofun-gin can be used for IFI treatment, thanks to their favorable pharmacodynamic/pharmacokinetic characteristics, lower tox-icity, and immunopharmacologic mode of action (4, 6, 9–12, 20, 23). This study aimed to compare the immunomodulating activities of caspofungin against Candida albicans on PMNs from RTRs and healthy subjects (HSs).
(This study was presented in part at the XLVII ERA-EDTA Congress, abstr. Sa283, Munich, Germany, 25 to 28 June 2010). All patients participating in this study gave their written informed consent. Blood samples were obtained from 30 HSs (controls) and from 54 RTRs, 31 males and 23 females (mean age, 53.9 years), without any evidence of infection, being fol-lowed at Ivrea Hospital (Turin, Italy). The mean time since transplantation was 83.94 months (range, 7 to 248 months; for 2/54 patients, less than 12 months since transplant), and the primary renal diseases which infected these patients were as follows: nephroangiosclerosis (n⫽ 16), chronic glomerulone-phritis (n⫽ 6), interstitial nephritis (n ⫽ 5), polycystic kidney disease (n⫽ 4), diabetic nephropathy (n ⫽ 4), chronic renal failure (n⫽ 10), and other (n ⫽ 9). The mean serum creatinine level at the time of the study was 1.7⫾ 0.8 mg/dl. Posttrans-plant immunosuppressive treatment used included the follow-ing: for the majority of patients, tacrolimus (FK), mycopheno-late mofetil (MMF), and prednisone (P); for 2 patients, cyclosporine (CyA) and MMF; for 7 patients, CyA, P, and
sirolimus; for 3 patients, P and FK; for 4 patients, FK and MMF; for 5 patients, FK alone; and for 1 patient, CyA alone. A clinical C. albicans strain, isolated from blood and iden-tified by biochemical methods, was subcultured on Sabouraud dextrose agar (Oxoid S.p.A., Milan, Italy) to ensure viability and purity (20). Caspofungin acetate (Merck Sharp & Dohme Ltd., Hoddesdon, United Kingdom) was dissolved in pyrogen-free water and stored at⫺20°C.
Antifungal susceptibility testing was performed with an in-oculum of 103CFU/ml, in accordance with CLSI M27-A3 (5),
and an inoculum of 106CFU/ml, used to perform tests with
phagocytes.
PMNs separated from lithium-heparinized venous blood us-ing Ficoll-Paque (Pharmacia S.p.A., Milan) were suspended in RPMI 1640 medium (106PMNs/ml) (Gibco Laboratories, NY)
as described previously (3). PMN viability was greater than 95%.
Both phagocytosis of radiolabeled C. albicans ([3H]uracil [specific activity, 1,270 GBq/mmol; NEN Life Science Prod-ucts, Milan]) and intracellular blastoconidia killing by PMNs were investigated by incubation of yeast cells (106yeast cells/
ml) and PMNs (1:1 ratio) at 37°C in a shaking water bath for 30, 60, and 90 min in the presence of one times the MIC (2 g/ml), one-half times the MIC, and one-fourth times the MIC of caspofungin. Drug-free controls were included. Phagocyto-sis and intracellular killing were assessed by the methods de-scribed previously (3, 20). Radioactivity was expressed as counts per minute (cpm) per sample. The percentage of phagocytosis at a given sampling time was calculated as fol-lows: percentage of phagocytosis⫽ [(number of cpm in PMN pellet)/(number of cpm in total fungal pellet)]⫻ 100 (3, 20). The PMN killing values were expressed as the survival index (SI), which was calculated by adding the number of surviving yeast cells at time zero to the number of survivors at time x and dividing by the number of survivors at time zero (3, 20). Ac-cording to this formula, if fungal killing was 100% effective, the SI would be 1.
Results were expressed as the means⫾ the standard errors
* Corresponding author. Mailing address: Department of Public Health and Microbiology, Microbiology Section, University of Turin, Via Santena 9, 10126 Turin, Italy. Phone: 39/0116705638. Fax: 39/ 0112365638. E-mail: [email protected].
䌤Published ahead of print on 3 October 2011.
5936
on November 18, 2011 by DIP DI SANITA PUBBLICA E MICROBIOLOGIA
http://aac.asm.org/
of the means (SEM) from 10 separate experiments, each per-formed in quadruplicate. Statistical evaluation of the differ-ences between test and control results was performed by Tukey’s test. A P value of⬍0.05 was considered significant.
RTR PMNs showed phagocytosis toward C. albicans similar to that registered for HS PMNs (Table 1), whereas they dis-played significant reduced fungicidal activity within 90 min (P⬍ 0.01), confirming our previous findings, which show that in RTRs, the number of PMNs was normal, but several met-abolic and functional alterations were observed, resulting in a high incidence of infections in these patients (19).
As observed for other systemic antifungal drugs (8, 16, 19, 20), no synergistic effect between PMNs and caspofungin on phagocytosis toward C. albicans in RTR or HS PMNs (Table 1) throughout the observation period occurred, indicating that caspofungin did not adversely interfere with PMN phagocytic capacity, probably due to a dramatic effect on the integrity of the yeast cell wall. The expression of surface molecules that link to cleavage products of complement could be altered with direct consequences for phagocytosis (8, 16, 20).
On the contrary, the addition of caspofungin significantly (P⬍ 0.01) elicited fungicidal activity against intracellular C.
albicans by HS PMNs compared to that observed in drug-free
controls (Table 1). Analogously, when caspofungin was added to RTR PMNs, their depressed fungicidal activity was com-pletely restored, with rates quite similar to those of HS PMNs, indicating that caspofungin synergized for fungal killing with PMNs being able to kill C. albicans at values significantly higher than those observed in drug-free RTR PMNs (P⬍ 0.01) (Table 1). A similar pattern was also detected when RTR PMNs and yeast cells were exposed to sub-MICs of caspofun-gin (data not shown), indicating the absence of a drug dose-dependent effect. The increased PMN killing activity observed at various caspofungin concentrations is related to a direct drug action on both C. albicans and phagocytes, as previously demonstrated (20) by the following: caspofungin is able both to unmask a fungal virulence factor in the yeast cell wall, making intracellular blastoconidia more susceptible to PMN lytic mechanisms, and to enter phagocytes remaining available in a biologically active form against proliferating yeast cells.
Taken together, the results of this study provide evidence that caspofungin is able to elicit the depressed phagocytic re-sponse of PMNs from RTRs, with the potential to totally
restore their primary functions in vitro. These findings support and could explain the improvement of survival in neutropenic high-risk patients with IFIs undergoing an empirical treatment with caspofungin versus liposomal amphotericin B, as de-scribed in the literature (17, 18, 24). In spite of the high cost of caspofungin, which nowadays limits its use to azole-resistant cases of candidal infections (22, 23), all these data permit the conclusion that caspofungin, in addition to its antifungal activ-ity, possesses immunomodulating properties that make it highly suitable for fungal infection treatment in patients with impaired components of the immune system, which represent a high-risk population.
To better assess these encouraging findings, further studies by testing both other Candida clinical isolates and other im-munocompromised patients are needed.
This work was supported by a grant from the Regione Piemonte, Ricerca Sanitaria Finalizzata (2008 bis), Italy.
We are grateful to the staff of both the Nephrology and Dialysis Unit (Ivrea Hospital, Ivrea, Turin, Italy) and Banca del Sangue e del Plasma (A.S.O. San Giovanni Battista, Turin, Italy) for their help in conduct-ing this study.
We have no conflicts of interest to declare.
REFERENCES
1. Anees, M. M., et al. 2010. Enhanced enzymatic activity of Candida species responsible for oral candidiasis in renal transplant recipients. Mycoses doi: 10.1111/j.1439-0507.2010.01874.x.
2. Badiee, P., P. Kordbacheh, A. Alborzi, and S. A. Malekhoseini. 2007. Inva-sive fungal infection in renal transplant recipients demonstrated by panfun-gal polymerase chain reaction. Exp. Clin. Transplant. 5:624–629. 3. Banche, G., et al. 2006. Effect of dialysis membrane biocompatibility on
polymorphonuclear granulocyte activity in dialysis patients. Nephrol. Dial. Transplant. 21:3532–3538.
4. Chiller, T., K. Farrokhshad, E. Brummer, and D. A. Stevens. 2001. The interaction of human monocytes, monocyte-derived macrophages, and poly-morphonuclear neutrophils with caspofungin (MK-0991), an echinocandin, for antifungal activity against Aspergillus fumigatus. Diagn. Microbiol. Infect. Dis. 39:99–103.
5. Clinical and Laboratory Standards Institute. 2008. Reference method for broth dilution antifungal susceptibility testing of yeasts, 3rd ed. Approved standard M27-A3. Clinical and Laboratory Standards Institute, Wayne, PA. 6. Deresinski, S. C., and D. A. Stevens. 2003. Caspofungin. Clin. Infect. Dis.
36:1445–1457.
7. Dongari-Bagtzoglou, A., et al. 2009. Oral Candida infection and colonization in solid organ transplant recipients. Oral Microbiol. Immunol. 24:249–254. 8. Frank, U., M. Greiner, I. Engels, and F. D. Daschner. 2004. Effects of
caspofungin (MK-0991) and anidulafungin (LY303366) on phagocytosis, oxidative burst and killing of Candida albicans by human phagocytes. Eur. J. Clin. Microbiol. Infect. Dis. 23:729–731.
9. Grover, N. D. 2010. Echinocandins: a ray of hope in antifungal drug therapy. Indian J. Pharmacol. 42:9–11.
TABLE 1. Effect of 1⫻ MIC of caspofungin against C. albicans on intracellular killing of PMNs from HSs and RTRs
Time (min)
Mean % phagocytosis⫾ SEM SI⫾ SEM (%)b
Controls PMNs treated with
caspofungin (2g/ml) Controls PMNs treated with caspofungin (2g/ml) HSs 30 77.1⫾ 0.67 77.2⫾ 0.76 1.54⫾ 0.03 (46) 1.29⫾ 0.07 (71)a 60 75.8⫾ 0.11 75.1⫾ 0.11 1.53⫾ 0.02 (47) 1.28⫾ 0.11 (72)a 90 75.1⫾ 0.32 75.9⫾ 0.09 1.52⫾ 0.03 (48) 1.25⫾ 0.07 (75)a RTRs 30 74.9⫾ 0.74 74.3⫾ 0.02 1.54⫾ 0.1 (46) 1.44⫾ 0.09 (56) 60 76.4⫾ 0.46 76.1⫾ 0.15 1.65⫾ 0.14 (35) 1.42⫾ 0.11 (58)a 90 76.4⫾ 0.58 76.7⫾ 0.31 1.74⫾ 0.08 (26) 1.33⫾ 0.11 (67)a a
Significantly different from controls (P⬍ 0.01).
b
Values shown in parentheses represent the percentages of yeast cells killed by PMNs in the absence and in the presence of the antifungal drug.
VOL. 55, 2011 CASPOFUNGIN AND RENAL TRANSPLANT RECIPIENT PMNs 5937
on November 18, 2011 by DIP DI SANITA PUBBLICA E MICROBIOLOGIA
http://aac.asm.org/
10. Gullo, A. 2009. Invasive fungal infections: the challenge continues. Drugs
69:65–73.
11. Hohl, T. M., M. Feldmesser, D. S. Perlin, and E. G. Pamer. 2008. Caspo-fungin modulates inflammatory responses to Aspergillus fumigatus through stage-specific effects on fungal beta-glucan exposure. J. Infect. Dis. 198:176– 185.
12. Lamaris, G. A., et al. 2008. Caspofungin-mediated beta-glucan unmasking and enhancement of human polymorphonuclear neutrophil activity against Aspergillus and non-Aspergillus hyphae. J. Infect. Dis. 198:186–192. 13. Matignon, M., et al. 2008. Outcome of renal transplantation in eight patients
with Candida sp. contamination of preservation fluid. Am. J. Transplant.
8:697–700.
14. Pappas, P. G., et al. 2010. Invasive fungal infections among organ transplant recipients: results of the Transplant-Associated Infection Surveillance Net-work (TRANSNET). Clin. Infect. Dis. 50:1101–1111.
15. Pourmand, G., et al. 2007. Infectious complications after kidney transplan-tation: a single-center experience. Transpl. Infect. Dis. 9:302–309. 16. Richardson, M. D., G. Scott, and G. S. Shankland. 1992. Effect of cilofungin
on phagocytosis and intracellular killing of Candida albicans by human neu-trophils. Eur. J. Clin. Microbiol. Infect. Dis. 11:22–26.
17. Ru¨ping, M. J., J. J. Vehreschild, and O. A. Cornely.2008. Patients at high
risk of invasive fungal infections: when and how to treat. Drugs 68:1941– 1962.
18. Ru¨ping, M. J., J. J. Vehreschild, and O. A. Cornely.2008. Antifungal treat-ment strategies in high risk patients. Mycoses 51:46–51.
19. Tullio, V., et al. 2003. Combined action of fluconazole and PMNs from uremic patients in clearing intracellular Candida albicans. J. Chemother.
15:301–303.
20. Tullio, V., et al. 2010. Synergy of caspofungin with human polymorphonu-clear granulocytes for killing Candida albicans. Antimicrob. Agents Che-mother. 54:3964–3966.
21. van Hal, S. J., et al. 2009. Candidemia following solid organ transplantation in the era of antifungal prophylaxis: the Australian experience. Transpl. Infect. Dis. 11:122–127.
22. Veroux, M., et al. 2007. Caspofungin in kidney transplant recipients with refractory invasive candidiasis. Nephrol. Dial. Transplant. 22:1487–1489. 23. Veroux, M., et al. 2006. Caspofungin in the treatment of azole-refractory
esophageal candidiasis in kidney transplant recipients. Transplant. Proc.
38:1037–1039.
24. Walsh, T. J., et al. 2004. Caspofungin versus liposomal amphotericin B for empirical antifungal therapy in patients with persistent fever and neutrope-nia. N. Engl. J. Med. 351:1391–1402.
5938 ALLIZOND ET AL. ANTIMICROB. AGENTSCHEMOTHER.
